With concerns about greenhouse gas emissions and uncertainty about the supply of oil, renewable biofuels have gained increasing attention. Bioethanol, the current major biofuel, is not an ideal replacement of gasoline because of its low energy density, high water solubility and high vapor pressure. Compared to ethanol, butanol is more hydrophobic, has a more similar energy content (27 MJ/L) to that of gasoline (32 MJ/L) and can be transported in the existing pipeline infrastructure. Thus, butanol is considered as a better drop-in biofuel than ethanol.
Butanol can be produced by anaerobic microorganisms such as Clostridium acetobutylicum and Clostridium beijerinckii in acetone-butanol-ethanol fermentation (ABE fermentation), which was once the second largest industrial fermentation in the world. In a typical ABE fermentation, butyrate and acetate are produced first, and then the culture undergoes a metabolic shift and solvents (butanol, acetone, and ethanol) are formed. Due to the complicated metabolic pathways involving acidogenesis and solventogenesis, plus spore-forming life cycle, ABE fermentation is difficult to control or manipulate. Furthermore, ABE fermentation usually suffers from low butanol yield (˜20% w/w), titer (<15 g/L) and productivity (<0.5 g/L·h) because of butanol toxicity and production of other byproducts including acetone, ethanol, acetate and butyrate. The low reactor productivity, butanol yield, and final butanol concentration make biobutanol from ABE fermentation uneconomical for the fuel market.
Since the first oil crisis in the early 1980's, there have been numerous attempts to improve butanol production via metabolic engineering of C. acetobutylicum and process engineering to alleviate inhibition caused by butanol and facilitate product recovery. Genetic engineering of C. acetobutylicum has failed to result in large increases in butanol yields and product titers to economically favorable levels. This is largely due to the fact that high concentrations of butanol are toxic to the bacteria that produce the solvent. To date, no genetically engineered C. acetobutylicum and related strains can meet the requirements for industrial use in butanol production.
There have been efforts to engineer non-solventogenic microbes, including E. coli, S. cerevisiae, B. subtilis, P. putida and L. brevis, for butanol production because of their potentially higher butanol tolerance and because they are easier to clone than Clostridia. Some microbes, including several strains of P. putida can tolerate up to 6% (w/v) butanol, although the butanol toxic threshold is between 1% and 2% (w/v) for most microorganisms. Most S. cerevisiae strains are tolerant to 1% (v/v) butanol with 60% relative specific growth rate, and three strains can grow in 2% (v/v) butanol with 10%-20% relative growth rate. For E. coli, its growth rate decreased to 20%-40% and 40%-60% at 1% (v/v) butanol at 37° C. and 30° C., respectively, and no growth was observed at 2% (v/v) butanol.
The Clostridia's solventogenic pathway, which involves multiple genes, has been cloned and expressed in several microorganisms. However, the highest butanol titer obtained so far was only 0.580 g/L in E. coli, 0.300 g/L in L. brevis, 0.120 g/L in P. putida, 0.024 g/L in B. subtilis, and 0.0025 g/L in S. cerevisiae. The slow and reversible turn-over rate of Clostridium butyryl dehydrogenase complex (bdh) could limit the butanol production. A keto-acid pathway has also been expressed in E. coli for n-butanol production, achieving butanol titer of 800 mg/L. Recently, a higher n-butanol titer of 4.65 g/L was obtained using a chimeric pathway assembled from three different organisms (R. eutrophus pha, C. acetobutylicum hbd, crt, and T. denticola ter replacing bdh) and overexpressing native pyruvate dehydrogenase complex (aceEF.Ipd) in E. coli. Furthermore, by increasing the availability of NADH, which is required for butanol biosynthesis, approximately 15 g/L of n-butanol was produced from glucose by the recombinant E. coli with an approximately 80% theoretical yield. Nevertheless, it has remained desirable to find non-solventogenic hosts that can produce n-butanol at a titer comparable to or higher than that from solventogenic Clostridia, which usually produce 12 g/L to 16 g/L butanol in ABE fermentation.